The present invention relates generally to compositions and methods useful for treating conditions associated with defects in cell proliferation, cell differentiation and/or cell survival. The invention is more particularly related to dual-specificity protein phosphatases, and polypeptide variants thereof. The present invention is also related to the use of such polypeptides to identify antibodies and other agents, including small molecules, that modulate signal transduction leading to proliferative responses, cell differentiation and/or cell survival.
Mitogen-activated protein kinases (MAP-kinases) are present as components of conserved cellular signal transduction pathways that have a variety of conserved members. MAP-kinases are activated by phosphorylation at a dual phosphorylation motif with the sequence Thr-X-Tyr (by MAP-kinase kinases), in which phosphorylation at the tyrosine and threonine residues is required for activity. Activated MAP-kinases phosphorylate several transduction targets, including transcription factors. Inactivation of MAP-kinases is mediated by dephosphorylation at this site by dual-specificity phosphatases referred to as MAP-kinase phosphatases. In higher eukaryotes, the physiological role of MAP-kinase signaling has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAP-kinase signaling, such as cancer.
Dual-specificity protein tyrosine phosphatases (dual-specificity phosphatases) are phosphatases that dephosphorylate both phosphotyrosine and phosphothreonine/serine residues (Walton et al., Ann. Rev. Biochem. 62:101-120, 1993). Several dual-specificity phosphatases that inactivate a MAP-kinase have been identified, including MKP-1 (WO 97/00315; Keyse and Emslie, Nature 59:644-647, 1992), MKP-2 (WO97/00315), MKP4, MKP-5, MKP-7, Hb5 (WO 97/06245), PAC1 (Ward et al.,Nature 367:651-654, 1994), HVH2 (Guan and Butch, J. Biol Chem. 270:7197-7203, 1995) and PYST1 (Groom et al., EMBO J. 15:3621-3632, 1996). Expression of certain dual-specificity phosphatases is induced by stress or mitogens, but others appear to be expressed constitutively in specific cell types. The regulation of dual-specificity phosphatase expression and activity is critical for control of MAP-kinase mediated cellular functions, including cell proliferation, cell differentiation and cell survival. For example, dual-specificity phosphatases may function as negative regulators of cell proliferation. It is likely that there are many such dual-specificity phosphatases, with varying specificity with regard to cell type or activation. However, the regulation of dual specificity phosphatases remains poorly understood and only a relatively small number of dual-specificity phosphatases have been identified.
Accordingly, there is a need in the art for an improved understanding of MAP-kinase signaling, and the regulation of dual-specificity phosphatases within MAP-kinase signaling cascades. An increased understanding of dual-specificity phosphatase regulation may facilitate the development of methods for modulating the activity of proteins involved in MAP-kinase cascades, and for treating conditions associated with such cascades. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods for identifying agents capable of modulating cellular proliferative responses. In one aspect, the present invention provides isolated DSP-15 polypeptides having the sequence of DSP-15 recited in SEQ ID NO:2, or a variant thereof that differs in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% of the residues in SEQ ID NO:2, such that the polypeptide retains the ability to dephosphorylate an activated MAP-kinase.
Within further aspects, the present invention provides an isolated polynucleotide that encodes at least ten consecutive amino acids of a polypeptide having a sequence corresponding to SEQ ID NO:2. In certain embodiments the invention provides an isolated polynucleotide that encodes at least fifteen consecutive amino acids of a polypeptide having a sequence corresponding to SEQ ID NO:2. Certain such polynucleotides encode a DSP-15 polypeptide. Still further, polynucleotides may be antisense polynucleotides that comprise at least 15 consecutive nucleotides complementary to a portion of a DSP-15 polynucleotide and/or that detectably hybridize to the complement of the sequence recited in SEQ ID NO:1 under conditions that include a wash in 0.1xc3x97SSC and 0.1% SDS at 50xc2x0 C. for 15 minutes. Also provided are expression vectors comprising any of the foregoing polynucleotides, and host cells transformed or transfected with such expression vectors.
The present invention further provides, within other aspects, methods for producing a DSP-15 polypeptide, comprising the steps of: (a) culturing a host cell as described above under conditions that permit expression of the DSP-15 polypeptide; and (b) isolating DSP-15 polypeptide from the host cell culture.
Also provided by the present invention are isolated antibodies, and antigen binding fragments thereof, that specifically bind to a DSP-15 polypeptide such as a polypeptide having the sequence of SEQ ID NO:2.
The present invention further provides, within other aspects, pharmaceutical compositions comprising a polypeptide, polynucleotide, antibody or fragment thereof as described above in combination with a physiologically acceptable carrier.
Within further aspects, the present invention provides methods for detecting DSP-15 expression in a sample, comprising: (a) contacting a sample with an antibody or an antigen-binding fragment thereof as described above, under conditions and for a time sufficient to allow formation of an antibody/DSP-15 complex; and (b) detecting the level of antibody/DSP-15 complex.
Within still other aspects, the present invention provides methods for detecting DSP-15 expression in a sample, comprising: (a) contacting a sample with an antisense polynucleotide as described above; and (b) detecting in the sample an amount of DSP-15 polynucleotide that hybridizes to the antisense polynucleotide. The amount of DSP-15 polynucleotide that hybridizes to the antisense polynucleotide may be determined, for example, using polymerase chain reaction or a hybridization assay.
The invention also provides DSP-15 polypeptides useful in screening assays for modulators of enzyme activity and/or substrate binding. Methods are also provided, within other aspects, for screening for an agent that modulates DSP-15 activity, comprising the steps of: (a) contacting a candidate agent with a DSP-15 polypeptide as described above, under conditions and for a time sufficient to permit interaction between the polypeptide and candidate agent; and (b) subsequently evaluating the ability of the polypeptide to dephosphorylate a DSP-15 substrate, relative to a predetermined ability of the polypeptide to dephosphorylate the DSP-15 substrate in the absence of candidate agent. Such methods may be performed in vitro or in a cellular environment (e.g., within an intact cell).
Within further aspects, methods are provided for screening for an agent that modulates DSP-15 activity, comprising the steps of: (a) contacting a candidate agent with a cell comprising a DSP-15 promoter operably linked to a polynucleotide encoding a detectable transcript or protein, under conditions and for a time sufficient to permit interaction between the promoter and candidate agent; and (b) subsequently evaluating the expression of the polynucleotide, relative to a predetermined level of expression in the absence of candidate agent.
Also provided are methods for modulating a proliferative response in a cell, comprising contacting a cell with an agent that modulates DSP-15 activity.
Within further aspects, methods are provided for modulating differentiation of a cell, comprising contacting a cell with an agent that modulates DSP-15 activity.
The present invention further provides methods for modulating cell survival, comprising contacting a cell with an agent that modulates DSP-15 activity.
Within related aspects, the present invention provides methods for treating a patient afflicted with a disorder associated with DSP-15 activity (or treatable by administration of DSP-15), comprising administering to a patient a therapeutically effective amount of an agent that modulates DSP-15 activity. Such disorders include Duchenne Muscular Dystrophy, as well as cancer, graft-versus-host disease, autoimmune diseases, allergies, metabolic diseases, abnormal cell growth, abnormal cell proliferation and cell cycle abnormalities.
Within further aspects, DSP-15 substrate trapping mutant polypeptides are provided. Such polypeptides differ from the sequence recited in SEQ ID NO:2 in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% of the residues in SEQ ID NO:2, such that the polypeptide binds to a substrate with an affinity that is not substantially diminished relative to DSP-15, and such that the ability of the polypeptide to dephosphorylate a substrate is reduced relative to DSP-15. Within certain specific embodiments, a substrate trapping mutant polypeptide contains a substitution at position 382 or position 413 of SEQ ID NO:2.
The present invention further provides, within other aspects, methods for screening a molecule for the ability to interact with DSP-15, comprising the steps of: (a) contacting a candidate molecule with a polypeptide as described above under conditions and for a time sufficient to permit the candidate molecule and polypeptide to interact; and (b) detecting the presence or absence of binding of the candidate molecule to the polypeptide. The step of detecting may comprise, for example, an affinity purification step, a yeast two hybrid screen or a screen of a phage display library.
In one aspect, the present invention provides isolated DSP-15 polypeptides comprising the sequence of DSP-15 alternate form recited in SEQ ID NO:21, or a variant thereof that differs in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% of the residues in SEQ ID NO:21, such that the polypeptide retains the ability to dephosphorylate an activated MAP-kinase.
Within further aspects, the present invention provides an isolated polynucleotide that encodes at least ten consecutive amino acids of a polypeptide having a sequence corresponding to SEQ ID NO:21. In certain embodiments the invention provides an isolated polynucleotide that encodes at least fifteen consecutive amino acids of a polypeptide having a sequence corresponding to SEQ ID NO:21. Certain such polynucleotides encode a DSP-15 alternate form polypeptide. Still further, polynucleotides may be antisense polynucleotides that comprise at least 15 consecutive nucleotides complementary to a portion of a DSP-15 alternate form polynucleotide and/or that detectably hybridize to the complement of the sequence recited in SEQ ID NO:20 under conditions that include a wash in 0.1xc3x97SSC and 0.1% SDS at 60xc2x0 C. for 15 minutes. Also provided are expression vectors comprising any of the foregoing polynucleotides, and host cells transformed or transfected with such expression vectors.
The present invention further provides, within other aspects, methods for producing a DSP-15 alternate form polypeptide, comprising the steps of: (a) culturing a host cell as described above under conditions that permit expression of the DSP-15 alternate form polypeptide; and (b) isolating DSP-15 alternate form polypeptide from the host cell culture.
Also provided by the present invention are isolated antibodies, and antigen binding fragments thereof, that specifically bind to a DSP-15 alternate form polypeptide such as a polypeptide having the sequence of SEQ ID NO:21.
The present invention further provides, within other aspects, pharmaceutical compositions comprising a polypeptide, polynucleotide, antibody or fragment thereof as described above in combination with a physiologically acceptable carrier.
Within further aspects, the present invention provides methods for detecting DSP-15 alternate form expression in a sample, comprising: (a) contacting a sample with an antibody or an antigen-binding fragment thereof as described above, under conditions and for a time sufficient to allow formation of an antibody/DSP-15 alternate form complex; and (b) detecting the level of antibody/DSP-15 alternate form complex.
Within still other aspects, the present invention provides methods for detecting DSP-15 alternate form expression in a sample, comprising: (a) contacting a sample with an antisense polynucleotide as described above; and (b) detecting in the sample an amount of DSP-15 alternate form polynucleotide that hybridizes to the antisense polynucleotide. The amount of DSP-15 alternate form polynucleotide that hybridizes to the antisense polynucleotide may be determined, for example, using polymerase chain reaction or a hybridization assay.
The invention also provides DSP-15 alternate form polypeptides useful in screening assays for modulators of enzyme activity and/or substrate binding. Methods are also provided, within other aspects, for screening for an agent that modulates DSP-15 alternate form activity, comprising the steps of: (a) contacting a candidate agent with a polypeptide as described above, under conditions and for a time sufficient to permit interaction between the polypeptide and candidate agent; and (b) subsequently evaluating the ability of the polypeptide to dephosphorylate a DSP-15 alternate form substrate, relative to a predetermined ability of the polypeptide to dephosphorylate the DSP-15 alternate form substrate in the absence of candidate agent. Such methods may be performed in vitro or in a cellular environment (e.g., within an intact cell).
Within further aspects, methods are provided for screening for an agent that modulates DSP-15 alternate form activity, comprising the steps of: (a) contacting a candidate agent with a cell comprising a DSP-15 alternate form promoter operably linked to a polynucleotide encoding a detectable transcript or protein, under conditions and for a time sufficient to permit interaction between the promoter and candidate agent; and (b) subsequently evaluating the expression of the polynucleotide, relative to a predetermined level of expression in the absence of candidate agent.
Also provided are methods for modulating a proliferative response in a cell, comprising contacting a cell with an agent that modulates DSP-15 alternate form activity.
Within further aspects, methods are provided for modulating differentiation of a cell, comprising contacting a cell with an agent that modulates DSP-15 alternate form activity.
The present invention further provides methods for modulating cell survival, comprising contacting a cell with an agent that modulates DSP-15 alternate form activity.
Within related aspects, the present invention provides methods for treating a patient afflicted with a disorder associated with DSP-15 alternate form activity (or treatable by administration of DSP-15 alternate form), comprising administering to a patient a therapeutically effective amount of an agent that modulates DSP-15 alternate form activity. Such disorders include cancer, graft-versus-host disease, autoimmune diseases, allergies, metabolic diseases, abnormal cell growth, abnormal cell proliferation and cell cycle abnormalities.
Within further aspects, DSP-15 alternate form substrate trapping mutant polypeptides are provided. Such polypeptides differ from the sequence recited in SEQ ID NO:21 in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% of the residues in SEQ ID NO:21, such that the polypeptide binds to a substrate with an affinity that is not substantially diminished relative to DSP-15 alternate form, and such that the ability of the polypeptide to dephosphorylate a substrate is reduced relative to DSP-15 alternate form. Within certain specific embodiments, a substrate trapping mutant polypeptide contains a substitution at position 382 or position 413 of SEQ ID NO:21.
The present invention further provides, within other aspects, methods for screening a molecule for the ability to interact with DSP-15 alternate form, comprising the steps of: (a) contacting a candidate molecule with a DSP-15 alternate form polypeptide or variant thereof as described above under conditions and for a time sufficient to permit the candidate molecule and polypeptide to interact; and (b) detecting the presence or absence of binding of the candidate molecule to the polypeptide. The step of detecting may comprise, for example, an affinity purification step, a yeast two hybrid screen or a screen of a phage display library.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.